Our ability to effectively treat malaria is threatened by increasingly widespread resistance to the limited number of frontline antimalarial drugs available. Therefore, new strategies for identifying novel chemical probes and/or therapeutic leads centered around biologically validated targets are critically needed. Here, we propose the integrated use of functional genetics and chemical biology approaches to enable more effective target-driven antimalarial drug discovery. The long-term goal is to take advantage of recent advances in malaria parasite genetics to qualify and prioritize previously unexplored biological targets and/or pathways for therapeutics discovery efforts. By focusing on this target category, we envision achieving the capability to discover chemical probes that better allow us to define fundamental biology and elucidate new options for antimalarial therapy, and ultimately identify effective therapeutic strategies against multidrug resistant parasites that are becoming increasingly prevalent and widespread. While target-based drug discovery is conceptually appealing, it has not been as successful as phenotypic screens in identifying approved antimalarial agents. However, given the substantial amount of genomics data and the improved functional genetics tools now available, more effective approaches for target-based discovery could dramatically improve this. The objectives of this research are to (1) establish an innovative approach for improving target-specific drug discovery while (2) seeking to determine potential probe/drug-like molecules that are selective against several prioritized target candidates. While it relatively is straightforward to discover small molecules with in vitro activity against a target, prioritizing compounds that truly act via that target to dictate the biological outcome has been challenging. Conversely, phenotypic screens immediately establish the biological efficacy of a given compound. However, identifying the target(s) through which these compounds act biologically is time consuming and often unsuccessful. In both cases, the ability to synergistically leverage empirical and rational (e.g. structure-based) approaches for lead compound optimization is adversely impacted. In this proposal, we integrate use of state-of-the-art functional genetic tools to encode target-specific information into phenotypic screens to facilitate rapid identification of compounds interacting with pre-specified targets. We seek to establish a generalized framework applicable to a broad range of targets varying in their proposed biological function, subcellular localization, biochemical and biophysical properties. We envision developing standardized assays and analytical pipelines to facilitate evaluation of various compound collections. The current proposal will focus on developing and validating low- to-moderate throughput assays. The proposed research is significant because it simultaneously leverages key strengths of using target-specific and phenotypic screens in malaria parasites into a rapid, robust and potentially scalable process that can contribute overall to improving the efficiency with which therapeutically valuable targets and lead compounds are advanced during preclinical translational efforts.